Human CD8 T-cell activation in acute and chronic chikungunya infection.

There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli.

BACKGROUND: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-ß1 and substrate stiffness affect fibroblast Fas expression are not well understood. METHODS: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-ß1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. RESULTS: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-ß1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-ß1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-ß1. CONCLUSIONS: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

Down-regulation of islet amyloid polypeptide expression induces death of human annulus fibrosus cells via mitochondrial and death receptor pathways.

Islet amyloid polypeptide (IAPP) exerts its biological effects by participating in the regulation of glucose metabolism and cell apoptosis. The main goal of the present study was to investigate the expression of IAPP in degenerated intervertebral disc tissue and IAPP's modulation of extracellular matrix (ECM) catabolic and anabolic genes in human AF cells. We found that the expression of IAPP, the calcitonin receptor, and receptor activity modifying protein decreased considerably in AF cells during the progression of intervertebral disc degeneration (IDD). Meanwhile, transfection with pLV-siIAPP decreased the expression of IAPP and its receptors and reduced glucose uptake and the expression of aggrecan, Col2A1, and BG. Down-regulation of IAPP also induced a significant increase in reactive oxygen species generation in AF cells, along with a decrease in matrix metalloproteinases and an increase in the concentration of cellular Ca2+, ultimately leading to death. Further analysis revealed that siIAPP intervention promoted the release of cytochrome c from mitochondria, resulting in the activation of Caspase-3 and Caspase-9. In contrast, significantly decreased expression of Caspase-3 and Caspase-9 was observed in AF cells transfected with pLV-IAPP. The concentrations of Fas and FasL proteins were significantly decreased in AF cells transfected with PLV-IAPP, while activation of the Fas/FasL system and cell death were induced by siIAPP intervention. Mechanistically, AMPK/Akt-mTOR signaling pathways were involved. In conclusion, down-regulation of IAPP expression induces the death of human AF cells via mitochondrial and death receptor pathways, potentially offering a novel therapeutic target for the treatment of IDD.

IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure.

Interferon gamma (IFN-γ) has been reported to have both negative and positive activity on hematopoietic cells, adding complexity to the interpretation of its pleiotropic functions. We examined the effects of IFN-γ on murine hematopoietic stem cells (HSCs) and progenitors in vitro and in vivo by using mouse models. IFN-γ treatment expanded bone marrow (BM) c-Kit(+)Sca1(+)Lin(-) (KSL) cell number but reduced BM KLCD150(+) and KLCD150(+)CD48(-) cells. IFN-γ-expanded KSL cells engrafted poorly when tested by competitive repopulation in vivo. KSL, KLCD150(+), and KLCD150(+)CD48(-) cells from IFN-γ-treated animals all showed significant upregulation in Fas expression. When cocultured with activated T cells in vitro, KSL and KLCD150(+) cells from IFN-γ-treated donors showed increased apoptosis relative to those from untreated animals, and infusion of activated CD8 T cells into IFN-γ-injected animals in vivo led to partial elimination of KSL cells. Exposure of BM cells or KSL cells to IFN-γ increased expression of Fas, caspases, and related proapoptotic genes and decreased expression of Ets-1 and other hematopoietic genes. In mouse models of BM failure, mice genetically deficient in IFN-γ receptor expression showed attenuation of immune-mediated marrow destruction, whereas effector lymphocytes from IFN-γ-deficient donors were much less potent in initiating BM damage. We conclude that the activity of IFN-γ on murine hematopoiesis is context dependent. IFN-γ-augmented apoptotic gene expression facilitates destruction of HSCs and progenitors in the presence of activated cytotoxic T cells, as occurs in human BM failure.

Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.

OBJECTIVES: Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: C57/BL6 mice weighing 28-30 g. INTERVENTIONS: Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. MEASUREMENTS AND MAIN RESULTS: Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. CONCLUSIONS: Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.

The receptor for the complement C3a anaphylatoxin (C3aR) provides host protection against Listeria monocytogenes-induced apoptosis.

Listeria monocytogenes is a Gram-positive intracellular bacterium that is acquired through tainted food and may lead to systemic infection and possible death. Despite the importance of the innate immune system in fighting L. monocytogenes infection, little is known about the role of complement and its activation products, including the potent C3a anaphylatoxin. In a model of systemic L. monocytogenes infection, we show that mice lacking the receptor for C3a (C3aR(-/-)) are significantly more sensitive to infection compared with wild-type mice, as demonstrated by decreased survival, increased bacterial burden, and increased damage to their livers and spleens. The inability of the C3aR(-/-) mice to clear the bacterial infection was not caused by defective macrophages or by a reduction in cytokines/chemokines known to be critical in the host response to L. monocytogenes, including IFN-γ and TNF-α. Instead, TUNEL staining, together with Fas, active caspase-3, and Bcl-2 expression data, indicates that the increased susceptibility of C3aR(-/-) mice to L. monocytogenes infection was largely caused by increased L. monocytogenes-induced apoptosis of myeloid and lymphoid cells in the spleen that are required for ultimate clearance of L. monocytogenes, including neutrophils, macrophages, dendritic cells, and T cells. These findings reveal an unexpected function of C3a/C3aR signaling during the host immune response that suppresses Fas expression and caspase-3 activity while increasing Bcl-2 expression, thereby providing protection to both myeloid and lymphoid cells against L. monocytogenes-induced apoptosis.

Functional analysis of miR-181a and Fas involved in hepatitis B virus-related hepatocellular carcinoma pathogenesis.

The hepatitis B virus (HBV) is responsible for most of hepatocellular carcinoma (HCC). However, whether HBV plays an important role during hepatocarcinogenesis through effecting miRNAs remains unknown. Here, we reported that HBV up-regulated microRNA-181a (miR-181a) by enhancing its promoter activity. Simultaneously, we found that miR-181a inhibited apoptosis in vitro and promoted tumor cell growth in vivo. TNF receptor superfamily member 6 (Fas) was further identified as a target of miR-181a. We also found that Fas could reverse the apoptosis-inhibition effect induced by miR-181a. Moreover, HBV could inhibit cell apoptosis by down-regulating Fas expression, which could be reversed by miR-181a inhibitor. Our data demonstrated that HBV suppressed apoptosis of hepatoma cells by up-regulating miR-181a expression and down-regulating Fas expression, which may provide a new understanding of the mechanism in HBV-related HCC pathogenesis.

Serine protease inhibitors interact with IFN-γ through up-regulation of FasR; a novel therapeutic strategy against cancer.

Among the many immunomodulatory and anti-tumor activities, IFN-γ up-regulates tumor cell death mediated by Fas receptor (FasR). Our and several other studies have demonstrated the involvement of trypsin-like proteases (TLPs) in the mode of action of IFN-γ. In the present study, we tried to unravel the role of serine proteases in IFN-γ induced Fas-mediated cell death. Our present results show that both tosyl-l-Lysine chloromethylketone (TLCK), a trypsin like protease inhibitor and tosyl-l-phenylalanine chloromethylketone (TPCK) - a chymotrypsin like protease (CLP) inhibitor, sensitize HeLa cells to Fas-mediated cell death. The combined effect of these protease inhibitors with anti-Fas was stronger than additive. In contrast, elastase inhibitor III (EI), which also contains the chloromethyl ketone moiety, was not active. Furthermore, co-addition of TLCK or TPCK with IFN-γ markedly enhanced Fas-induced cell death. IFN-γ led to up-regulation of FasR on its own, which was further enhanced by the co-addition of TLCK or TPCK. This was evident both by increased expression of Fas receptor on cell surface and by elevated Fas mRNA level. This study may provide the basis for the design of a novel combinatory therapeutic strategy that could enhance the eradication of tumors.

ING4 Inhibits Proliferation and Induces Apoptosis in Human Melanoma A375 Cells via the Fas/Caspase-8 Apoptosis Pathway.

BACKGROUND: Inhibitor of growth 4 (ING4) plays a role in regulating the cell cycle, apoptosis, cell invasion and migration, but the mechanisms involved remain to be elucidated. OBJECTIVE: To explore how ING4 affects human malignant melanoma A375 cells. METHODS: Recombinant lentiviral vectors (A375/pLenO-GTP-ING4) were constructed and transfected into A375 cells (experimental group). The impact of ING4 on the proliferation and apoptosis of A375 cells was investigated in in vitro and in vivo experiments in mice using the MTT assay and flow cytometry. RESULTS: In the experimental group, optical density was lower and apoptotic cells were more frequent from days 2-5 (p = 0.000 and p < 0.01); there were smaller xenografts and more apoptotic cells in mice (all p < 0.05); moreover, increased levels of Fas, cleaved caspase-8 and caspase-3, and decreased levels of FasL and procyclic acidic repetitive protein were observed in vitro and in vivo. CONCLUSION: ING4 might suppress proliferation and enhance apoptosis in human malignant melanoma cells by activating Fas-induced apoptosis in a caspase-8-dependent pathway.

Supplementation freeze-thawed media with selenium protect adipose-derived mesenchymal stem cells from freeze-thawed induced injury.

Successful freezed-thaw of adipose-derived mesenchymal stem cells (ADMSCs) could be a major step in regenerative medicine as well as in the cloning of animal breeds. The aim of this study was to evaluate the efficacy of selenium on the optimizing of freezed-thaw media in the ADMSCs. ADMSCs were extracted from NMRI mice and purified with positive selection Monoclonal CD105 Antibody (PE) and negative selection Monoclonal CD31 and CD45 Antibody using MACS method as well as differentiation to adipose and bone tissue. ADMSCs were divided into four groups. ADMSCs were freezed-thaw under standard condition with or without the addition of 5 ng/ml selenium to both the cryopreservation and thawing solutions. Frozen cells were thawed after four months and viability and cytotoxicity of the cells were analyzed by the Trypan blue test and MTT assay respectively. RNA was extracted and cDNA was synthesized and the expression of apoptotic genes (P53, Fas, Bax, Caspase3, and Bcl2) was examined using Real time-PCR Rotor gene 2009. This study compares slow and rapid methods of cryopreservation. After thawing, viability of the cells treated with selenium was higher than the control group in rapid and slow cryopreserved ADMSCs. Also, the percentage of living cells in the slow cooling method was considerably more than with the rapid cooling method. After analysis of the results using Real time-PCR, the Bcl2 gene was shown to be expressed in both the rapid and slow cooling methods. In the rapid cooling group in addition to the BCL-2 gene, p53 was also expressed. It appears that selenium prevented the apoptotic genes from expression due to its anti-apoptotic effects. The slow cooling method is better and more optimized for ADMSCs protecting them from oxidative damage to a greater extent compared to the rapid cooling method.

Expression of miR-98 in myocarditis and its influence on transcription of the FAS/FASL gene pair.

Myocarditis is a common cardiovascular disease and frequently occurs in children and teenagers. It is believed to be caused by both endogenous and exogenous factors, among which FAS/FASL gene pair-induced cell apoptosis is a major mechanism of myocardial cell injury. A previous study has detected low expression of microRNA (miR)-98 in myocarditis patients. Therefore, in this study we investigated the functional implications of miR-98 with respect to the disease. We carried out a case-control study including 50 myocarditis patients and 50 healthy individuals. Total RNA was extracted from peripheral blood plasma. Expression levels of miR-98 and the FAS/FASL gene pair were determined by real-time fluorescent quantitative polymerase chain reaction. The interaction between miR-98 and the FAS/FASL pair was visualized by dual-luciferase reporter assay. The expression of the FAS/FASL gene pair was further detected by transfecting with an miR-98 mimic or an miR-98 inhibitor. The content of miR-98 in the peripheral blood of the myocarditis patients was significantly lower than in the healthy individuals. However, the FAS/FASL genes were upregulated by 1.68-fold in the myocarditis patients. miR-98 was shown to interact with the 3'-untranslated region of the FAS/FASL gene pair. The inhibition/facilitation of miR-98 expression in myocardial cells can modulate apoptosis. miR-98 was downregulated in the peripheral blood of myocarditis patients. It may interact with the FAS/FASL gene pair to further modulate cell apoptosis.

Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression.

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.

Epigenetic regulation of microRNA-128a expression contributes to the apoptosis-resistance of human T-cell leukaemia jurkat cells by modulating expression of fas-associated protein with death domain (FADD).

Increased expression of miR-128a is often observed in acute lymphoblastic leukaemia (ALL) compared with its expression in acute myeloid leukaemia (AML). The objective of this study was to investigate the role of miR-128a, especially that in the Fas-signalling pathway, in T-cell leukaemia cells. The role of miR-128a in Fas-mediated apoptosis was examined by using Fas-activating antibody (CH-11)-susceptible Jurkat cells and -resistant Jurkat/R cells. Whereas ectopic expression of miR-128a conferred Fas-resistance on Jurkat cells by directly targeting Fas-associated protein with death domain (FADD), antagonizing miR-128a expression sensitized Jurkat/R cells to the Fas-mediated apoptosis through derepression of FADD expression. Myeloid leukaemia HL60 and K562 cells were also CH-11-resistant, sharing a similar resistant mechanism with Jurkat/R cells. Furthermore, CH-11 induced demethylation of the promoter region of miR-128a with resultant up-regulation of miR-128a expression in Jurkat/R cells, which was shown to be a mechanism for the resistance ofJurkat/R cells to Fas-mediated apoptosis. Our results indicate that the induction of miR-128a expression by DNA demethylation is a novel mechanism of resistance to Fas-mediated apoptosis.

Paclitaxel-induced transcriptional regulation of Fas signaling pathway is antagonized by dexamethasone.

Chemotherapy agents like paclitaxel are often a first line of therapy for triple-negative breast cancer patients and a last line of therapy for other aggressive breast cancers. While such agents are effective pro-apoptotic treatments for breast cancer cells, they produce many unwanted side effects. Synthetic glucocorticoids like dexamethasone are commonly prescribed during chemotherapy treatment of breast cancer patients for anti-emetic and anti-hypersensitivity purposes. Dexamethasone, however, is known to enhance cell survival in solid tumors. The prevalent use of dexamethasone in conjunction with paclitaxel in the treatment of breast cancer patients merits concern. Previous studies demonstrate dexamethasone-mediated survival in a subset of breast cancer cells in the presence of paclitaxel. This work expanded the types of cell lines studied and explored the molecular mechanism driving this phenotype. Human breast cancer cell lines representative of several subtypes of breast cancer including triple-negative breast cancer, luminal, and HER2 were treated in the presence and absence of paclitaxel, dexamethasone, and under co-treatment conditions. Cell survival was monitored under differing conditions. We then manipulated aspects of the nuclear factor kappa B (NFkappaB) signaling pathway to elucidate the importance of this pathway in overcoming chemotherapy treatment. All cell lines tested were sensitive to paclitaxel and showed dexamethasone-mediated rescue from paclitaxel-induced apoptosis, but the rescue of overall cell numbers was most pronounced in triple-negative breast cancer cell lines. Dexamethasone and paclitaxel inversely regulated the activity of NFkappaB, which is essential to both paclitaxel-mediated apoptosis and dexamethasone-mediated rescue. The transcriptional target of NFkappaB, Fas receptor, is inversely regulated by paclitaxel and dexamethasone and is a downstream target of paclitaxel-activated NFkappaB. Dexamethasone antagonizes paclitaxel-mediated apoptosis through inhibition of NFkappaB transcription of Fas receptor. Pre-treatment of breast cancer patients with dexamethasone may greatly reduce patient response to paclitaxel. Our study elucidates a novel mechanism of paclitaxel-induced apoptosis in breast cancer cell lines and explicates dexamethasone's antagonism of paclitaxel.

Forced miR-146a expression causes autoimmune lymphoproliferative syndrome in mice via downregulation of Fas in germinal center B cells.

By inhibiting target gene expression, microRNAs (miRNAs) play major roles in various physiological and pathological processes. miR-146a, a miRNA induced upon lipopolysaccharide (LPS) stimulation and virus infection, is also highly expressed in patients with immune disorders such as rheumatoid arthritis, Sjögren's syndrome, and psoriasis. Whether the high level of miR-146a contributes to any of these pathogenesis-related processes remains unknown. To elucidate the function of miR-146a in vivo, we generated a transgenic (TG) mouse line overexpressing miR-146a. Starting at an early age, these TG mice developed spontaneous immune disorders that mimicked human autoimmune lymphoproliferative syndrome (ALPS) with distinct manifestations, including enlarged spleens and lymph nodes, inflammatory infiltration in the livers and lungs, increased levels of double-negative T cells in peripheral blood, and increased serum immunoglobulin G levels. Moreover, with the adoptive transfer approach, we found that the B-cell population was the major etiological factor and that the expression of Fas, a direct target of miR-146a, was significantly dampened in TG germinal center B cells. These results indicate that miR-146a may be involved in the pathogenesis of ALPS by targeting Fas and may therefore serve as a novel therapeutic target.

Fas-mediated inflammatory response in Listeria monocytogenes infection.

The molecular mechanisms of Fas (CD95/Apo-1)-mediated apoptosis are increasingly understood. However, the role of Fas-mediated production of proinflammatory cytokines such as IL-18 and IL-1ß in bacterial infection is unclear. We demonstrate the importance of Fas-mediated signaling in IL-18/IL-1ß production postinfection with Listeria monocytogenes without the contribution of caspase-1 inflammasome. IL-18/IL-1ß production in L. monocytogenes-infected peritoneal exudate cells from Fas-deficient mice was lower than those from wild type mice, indicating that Fas signaling contributes to cytokine production. L. monocytogenes infection induced Fas ligand expression on NK cells, which stimulates Fas expressed on the infected macrophages, leading to the production of IL-18/IL-1ß. This was independent of caspase-1, caspase-11, and nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) such as Nlrp3 and Nlrc4, but dependent on apoptosis-associated speck-like protein containing a caspase recruitment domain. Wild type cells exhibited caspase-8 activation, whereas Fas-deficient cells did not. L. monocytogenes-induced caspase-8 activation was abrogated by inhibitor for intracellular reactive oxygen species, N-acetyl-L-cysteine. L. monocytogenes-infected macrophages produced type-I IFNs such as IFN-ß1, which was required for Il18 gene expression. Thus, Fas signaling regulates innate inflammatory cytokine production in L. monocytogenes infection.

Sevoflurane induces neurotoxicity in young mice through FAS/FASL signaling.

Sevoflurane, the most widely used anesthetic in clinical practice, has been shown to induce apoptosis, inhibit neurogenesis, and cause learning and memory impairment in young mice. However, the underlying mechanism is still unknown. In this study, wild-type and the FAS- or FAS ligand (FASL)-knockout mice (age 7 days) were exposed to sevoflurane or pure oxygen. Western blotting was used to examine the expression of FAS protein. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and bromodeoxyuridine (BrdU) staining were employed to quantify the apoptotic cells and newborn cells in the hippocampus and Morris water maze (MWM) in order to evaluate learning and memory status. Sevoflurane significantly increased the expression of FAS protein in wild-type mice. Compared to FAS- and FASL-knockout mice treated with sevoflurane, sevoflurane-treated wild-type mice exhibited more TUNEL-positive hippocampal cells and less BrdU-positive hippocampal cells. The MWM showed that compared with FAS- and FASL-knockout mice treated with sevoflurane, sevoflurane treatment of wild-type mice significantly prolonged the escape latency and reduced platform crossing times. These data suggest that sevoflurane induces neurotoxicity in young mice through FAS-FASL signaling.

[The Changes of Properties of Blood Peripheral Lymphocytes of Donors and Patients with Prostate Gland Cancer: Reaction of Lymphocytes on Irradiation in vitro].

The oxidative status (ROS), markers activation expression (CD69), proliferation activity (Ki67), proapoptotic antigen (CD95) have been investigated on healthy donors and patients with prostatic gland cancer in human blood lymphocytes. The lymphocyte reaction in vitro on γ-irradiation at different doses (0.05-1.0 Gy) has been determined too. It was shown that in these two types of individuals the ROS content does not differ and the reaction on irradiation is not different either. Essential is the difference between the marker expression in lymphocytes of healthy donors and patients with tumour: in individuals with cancer the content of lymphocytes with CD69+ phenotype (in non active situation) and CD95+ increases, the expression of marker Ki67 decreases. The lymphocyte response to irradiation in healthy and tumour lymphocytes is distinguished. Irradiation at doses 0.05-10.0 Gy on tumour patients lymphocytes markers does not influence expression. In healthy donors' lymphocytes the expression of markers is changed considerably, the reaction depends on the marker type: expression of CD69 marker decreases (tendency); expression of Ki67 decreases too; it is unusual that the expression of CD95 changes--it decreases after irradiation at the doses of 0.05-1.0 Gy, then increases with dose. So this work shows the changes in tumour patients' blood lymphocytes in comparison with healthy donors' lymphocytes. The possible mechanisms of the observed phenomenon are discussed.

Tumor-induced myeloid-derived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8+ T-cell activation events.

Tumor growth coincides with an accumulation of myeloid-derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b(+) CD115(+) Ly6G(-) Ly6C(high) MDSCs and granulocytic CD11b(+) CD115(-) Ly6G(+) Ly6C(int) polymorphonuclear (PMN)-MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8(+) T-cell activation--including cytokine production, surface marker expression, survival, and cytotoxicity--is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen-driven CD8(+) T-cell proliferation, but differ in their dependency on IFN-γ, STAT-1, IRF-1, and NO to do so. Moreover, MO-MDSC and PMN-MDSCs diminish IL-2 levels, but only MO-MDSCs affect IL-2Rα (CD25) expression and STAT-5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN-γ production by CD8(+) T cells on a per cell basis, illustrating that some T-cell activation characteristics are actually stimulated by MDSCs. Conversely, MO-MDSCs counteract the activation-induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8(+) T-cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL-mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8(+) T-cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.

Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.

MHC class Ia-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.